In the context of virology, transfer RNA (tRNA) has been known to function as a primer for retroviral reverse transcriptases while the tRNA-like structural elements (TLS) found at the 3' end of the genomes of many plant RNA viruses serve as efficient origins of replications. Our recent in vitro assembly assays with RNA transcripts and coat protein subunits of an icosahedral plant RNA virus, brome mosaic virus (BMV), revealed an unprecedented function for tRNAs (viral and non-viral origins) in the life cycle of the virus, namely virus assembly. The overall aim of this proposal is to develop our understanding of the key role-played by tRNAs in virus assembly. Our proposed research will describe in detail the extent to which tRNAs are involved in promoting assembly of viral RNAs into icosehedral virions. In part, this study will involve evaluating the trans-acting role of host tRNAs in virus assembly using a novel Agrobacterium-mediated transient assay in a replication independent mode. Additional proposed experiments will characterize the biochemical nature of intermediate structures formed between tRNAs and coat protein (CP) subunits in the assembly pathway of icosahedral virions by exclusion chromatography and electron microscopy. In BMV, genomic RNA3 (B3) and CP subgenomic RNA4 (B4) co-package into a single virion. A second part of the investigation will identify and characterize the packaging signal localized on the dicistronic B3 with respect to structure and functionality. The mechanism by which B3 and B4 are co-packaged will be examined in vitro and in vivo. For evaluating sequential packaging mechanism, we propose three independent approaches: (i) a two-step in vitro assembly assay will evaluate whether prior packaging of B3 is a prerequisite for B4 co-packaging; (ii) the application of MALDI-MS to pre-assembled virions of B3 with wild type and mutant CP will examine the surface organization; (iii) Agrobacterium-mediated transient assays will aid in confirming the in vitro data in vivo. Additional biochemical experiments are proposed to evaluate whether co-packaging of B3 and B4 occurs via RNA-RNA interactions mediated through an alternate concerted packaging mechanism. Collectively, the proposed studies will advance our understanding of the assembly mechanisms of icosahedral viruses that are known to cause economically important diseases in humans, animals, insects and plants.